A new pathway of nitric oxide/cyclic GMP signaling involving S-nitrosoglutathione.
Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from L-arginine and O2 in a reaction catalyzed by NO synthases (NOS). Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O-2). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3',5'-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 +/- 0.01 and 0.19 +/- 0.06 micromol of cGMP x mg-1 x min-1 without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner. Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 +/- 0.60 micromol of cGMP x mg-1 x min-1). The NO/O-2 donor SIN-1 caused only a slight accumulation of cGMP in the absence of GSH but was almost as effective as DEA/NO in the presence of the thiol. The profile of sGC activation by Ca2+/calmodulin-activated NOS resembled that of SIN-1; at a maximally active concentration of 200 ng/0.1 ml, NOS increased sGC activity to 1.22 +/- 0.12 and 8.51 +/- 0.88 micromol of cGMP x mg-1 x min-1 in the absence and presence of GSH, respectively. The product of NOS and GSH was identified as the thionitrite GSNO, which activated sGC through Cu+-catalyzed release of free NO. In contrast to S-nitrosation by peroxynitrite, the novel NO/O-2-triggered pathway was very efficient (25-45% GSNO) and insensitive to CO2. Cu+-specific chelators inhibited bradykinin-induced cGMP release from rat isolated hearts but did not interfere with the direct activation of cardiac sGC, suggesting that thionitrites may occur as intermediates of NO/cGMP signaling in mammalian tissues.
Mayer
B
B
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria. mayer@kfunigraz.ac.at
Pfeiffer
S
S
Schrammel
A
A
Koesling
D
D
Schmidt
K
K
Brunner
F
F
D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism*; D004789:Enzyme Activation; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism; D066298:In Vitro Techniques; D009206:Myocardium / Q000378:metabolism; D009474:Neurons / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009603:Nitroso Compounds / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D026422:S-Nitrosoglutathione; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000378:metabolism
D009603:Nitroso Compounds; D011994:Recombinant Proteins; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D006162:Guanylate Cyclase; D005978:Glutathione; D006152:Cyclic GMP